Abstract

1. Introduction

The Coronaviridae are a family of enveloped, positive-strand RNA viruses that have a broad host range, although they are predominantly associated with mammals and birds. Several coronaviruses have emerged as highly pathogenic viruses in humans and other animals, the most recent and notable of which is severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) (Wu et al. 2020), which, at the time of writing, has infected over 150 million people since its initial identification at the end of 2019. Coronaviruses, like other families within the order Nidovirales, possess the longest genomes seen in RNA viruses (27–34 kb in the case of the coronaviruses) and seemingly replicate with higher fidelity than other known positive-sense RNA viruses due to RNA proofreading associated with an exonuclease domain (Denison et al. 2011).

Most coronaviruses are classified within the sub-family Orthocoronavirinae that infect mammals and birds as well as a single virus identified in a reptilian host (Shi et al. 2018). However, our understanding of the host range of the Coronaviridae has recently been enhanced by large-scale metagenomic studies, including the identification of a distinct second sub-family: the Letovirinae (Coronaviridae Study Group of the International Committee on Taxonomy of Viruses, Gorbalenya et al. 2020). Specifically, an analysis of published transcriptome data identified a novel coronavirus in the ornamented pygmy frog (Microhyla fissipes) termed Microhyla letovirus (Bukhari et al. 2018). A subsequent virological survey of dead and moribund cultured Chinook salmon (Oncorhynchus tshawytscha) identified Pacific salmon nidovirus (Mordecai et al. 2019). Phylogenetic analysis revealed that Microhyla letovirus and Pacific salmon nidovirus formed a sister group to all other known coronaviruses, with the large genetic divergence between them suggesting that this group is likely far larger and under-sampled (Bukhari et al. 2018; Mordecai et al. 2019). It is therefore unsurprising that more recent probes of public sequencing data have identified additional coronavirus-like sequences in non-mammalian aquatic vertebrate hosts, namely in fish and amphibian transcriptomes (Edgar et al. 2020). Indeed, this extensive data mining study identified six novel viruses within the Letovirinae: five in bony fish and one in the axolotl (Ambystoma mexicanum) (Edgar et al. 2020). More recently, an additional virus identified in the common carp (Cyprinus carpio) in Australia was also found to fall within this group (Costa et al. 2021).

The central aim of the current study was to determine whether coronaviruses might be present in a wider range of fish hosts and from this reveal more of their evolutionary history, particularly whether coronaviruses have co-diverged with their hosts over many millions of years or whether there has been frequent cross-species transmission (i.e. host jumping) as underpins the emergence of SARS-CoV-2 and whether such cross-species transmission occurs in some hosts more frequently than others.

To this end, we screened for coronaviruses using total RNA sequencing (i.e. meta-transcriptomic) data from jawless and bony fish. Jawless fish included the pouched lamprey (Geotria australis) sampled from New Zealand—te reo Māori: kanakana (South Island) or piharau (North Island)—that displayed lamprey reddening syndrome (LRS). This syndrome is characterised by red lesions on their gills, fins, and body walls (Brosnahan et al. 2019), but a causative agent has yet to be identified. We combined this investigation with data mining of published RNA sequencing data sampled from fish within the National Centre for Biotechnology Information (NCBI) Sequence Read Archive (SRA). This enabled us to identify new coronavirus sequences that not only significantly expanded the phylogenetic diversity of the coronaviruses but that provided important new insights into the fundamental patterns and processes of viral evolution in this virus family.

2. Results

2.1. Identification of a novel coronavirus in the pouched lamprey

Following a disease investigation into LRS in pouched lamprey from New Zealand by the New Zealand Ministry for Primary Industries (MPI) in 2012, 12 individuals (eight from this investigation and four more recently collected control samples) were subjected to meta-transcriptomic analysis. This led to the identification of a novel and divergent member of the Coronaviridae in several individuals. We have named this new virus kanakana letovirus, and as each virus shared >97per cent sequence similarity at the nucleotide level, they are likely genomic variants of the same virus species.

We detected sequence reads that shared sequence identity to coronaviruses in seven of the eight LRS-affected individuals (confirmed by reverse transcription-polymerase chain reaction (RT-PCR)). No coronaviruses or other viruses were found in the remaining LRS sample nor the four control samples. However, we were only able to generate consensus virus sequences from three of these due to the low and fragmented virus abundance in the other samples. All three samples possessed virus transcripts with sequence similarity to the open reading frames 1a (ORF1a) and 1b (ORF1b) common to the Coronaviridae, the latter of which contains the viral RNA-dependent RNA polymerase (RdRp) (Table 1). We also identified transcripts with marked sequence similarity to the Nidovirales spike (S) protein. However, the closest sequence match for the S protein transcripts were viruses from Porcine torovirus and Atlantic salmon bafinivirus, both non-coronavirus members of the Nidovirales (family Tobaniviridae), albeit these were highly divergent sequences with only ~25–27per cent amino acid sequence similarity (Table 1). Phylogenetic comparisons of the ORF1ab and S genomic regions of kanakana letovirus in the context of its closest relatives illustrated this striking incongruence (Fig. 1). In particular, both Microhyla letovirus and kanakana letovirus were more closely related to coronaviruses in the ORF1ab region but not in the S protein where they exhibited greater sequence similarity to the Tobaniviridae, suggestive of past recombination events (Fig. 1). In contrast, Pacific salmon nidovirus consistently fell as a sister group to the Coronaviridae in both gene regions, although with long branch lengths indicative of long periods of evolutionary divergence (Fig. 1).

Table 1.

Novel virus transcripts identified in this study from pouched lamprey (Geotria australis), their corresponding closest genetic match on GenBank, and standardised abundances.

Virus	LRS symptoms	Host	Gene	Sequence length (nt)	Closest match on GenBank (accession); e-value	Sequence identity (%)	Standardised viral abundance	RSP13 abundance
Kanakana letovirus (1)	Light haemorrhaging around gill slits.	Geotria australis	ORF1a	9,270	Pacific salmon nidovirus ORF1a (QEG08236.1); e-value=1 × 10−155	26.36	6.86 × 10−5	5.52 × 10−6
			ORF1b	6,507	Pacific salmon nidovirus ORF1b (QEG08237.1); e-value=0	46.53	4.67 × 10−5
			Spike	5,022	Porcine torovirus spike protein (QBJ02013.1); e-value=2 × 10−10	27	4.24 × 10−5
Kanakana letovirus (2)	Small area of haemorrhaging near the ventral fin.	Geotria australis	ORF1a	9,255	Pacific salmon nidovirus ORF1a (QEG08236.1); e-value=5 × 10−49	27.81	1.48 × 10−5	6.75 × 10−6
			ORF1b	6,501	Pacific salmon nidovirus ORF1b (QEG08237.1); e-value=4 × 10−175	46.30	1.26 × 10−5
			Spike	4,989	Atlantic salmon bafinivirus spike protein (AUE23862.1); e-value=2 × 10−4	24.07	6.4 × 10−6
Kanakana letovirus (3)	Haemorrhaging on dorsal fins and gill slits. Pale liver with haemorrhaging, swollen kidney, adhesions between organs and muscles.	Geotria australis	ORF1a	9,270	Pacific salmon nidovirus ORF1a (QEG08236.1); e-value=2 × 10−156	26.22	6.38 × 10−5	6.40 × 10−6
			ORF1b	6,509	Pacific salmon nidovirus ORF1b (QEG08237.1); e-value=0	43.97	4.20 × 10−5
			Spike	5,022	Porcine torovirus spike protein (QBJ01983.1); e-value=1 × 10−9	27.18	4.48 × 10−5

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Figure 1.

Unrooted phylogenetic trees for the open reading frame 1ab region (blue) and the spike protein (pink). Branches for viruses classified within the Tobaniviridae are shown in black while all other branches are coloured. A dashed line circles those taxa that are closest known relatives of kanakana letovirus in each case. Four of the viruses discovered in this study in which spike proteins were identified are in bold. All branches are scaled according to the number of amino acid substitutions per site. Percentage node labels with bootstrap support >70per cent are indicated with an asterisk (*). Supplementary Table S2 provides the accession numbers for each sequence used in the trees. Amino acid alignments are available at https://github.com/jemmageoghegan/Fish_coronaviridae.

All three kanakana letovirus sequencing libraries had similar sequencing depths as illustrated by the standardised abundance of the host gene, RSP13, which was detected at a lower abundance than kanakana letovirus (Fig. 2). However, the kanakana letovirus in samples 1 and 3 were the most abundant across the ORF1ab and S proteins. The presence of kanakana letovirus in seven of the individual lampreys was confirmed by RT-PCR (Supplementary Fig. S1). No other viruses were detected in these data. An amino acid sequence comparison with Microhyla letovirus across all three genomic regions shows relatively high sequence similarity in ORF1b (Fig. 2).

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Figure 2.

(A) Standardised abundance of kanakana letovirus transcripts (yellow) in comparison to the lamprey host gene, RSP13 (green), across the three lamprey samples that contained the highest abundance of the novel coronavirus. (B) Schematic alignment of all three transcripts against Microhyla letovirus, along with the percentage sequence identity.

2.3. The evolutionary history of novel coronaviruses in aquatic vertebrates

We next inferred the evolutionary relationships of all the viruses newly identified here and related Nidovirales through phylogenetic analysis of amino acid sequences of ORF1b that includes the highly conserved RdRp. As noted above, two viruses identified here fell within the Tobaniviridae, rather than the Coronaviridae, and were close genetic relatives of other fish bafiniviruses (Cano et al. 2020) (Fig. 3B). This analysis also revealed that fish coronaviruses fell only within the subfamily Letovirinae and were distinct from the Orthocoronavirinae. Specifically, the hosts of viruses in the Letovirinae comprised only aquatic vertebrates: both jawless and bony fish as well as viruses from amphibians (Fig. 3C).

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Figure 3.

(A) Schematic phylogram showing where bony and jawless fish fall in the greater animal phylogeny and colour-coded to signify the host class in (B) and (C), informed by the relevant literature on vertebrate evolution. (B) An order-level phylogenetic tree showing the relationships between families within the Nidovirales. Branches are colour-coded to signify the host class shown in part a. Branches in bold represent those viruses identified in this study. All branches are scaled according to the number of amino acid substitutions per site. Supplementary Table S3 provides the accession numbers for each sequence used in the tree. Amino acid alignments are available at https://github.com/jemmageoghegan/Fish_coronaviridae. (C) Maximum likelihood phylogenetic tree of the open reading frame 1b (ORF1b), containing the viral RdRp, showing the topology of the nine newly discovered coronaviruses (bold font; blue and cyan) that fall within Letovirinae. The order-level tree in (B) was used to root the tree. All branches are scaled according to the number of amino acid substitutions per site. Percentage node labels with bootstrap support >70per cent are indicated with an asterisk (*).

Although there are broad patterns of host clustering across the coronaviruses as a whole with, for example, most of the mammalian coronaviruses falling in the genera Alphacoronavirus and Betacoronavirus, and the genus Deltacoronavirus comprising avian-associated viruses only, it is notable that the phylogenetic position of the Letovirinae does not follow that of their hosts (Fig. 4A). Of particular note, kanakana letovirus sampled from New Zealand pouched lamprey falls as are a relative ingroup in this subfamily (Fig. 3). Similar patterns can be seen for viruses in amphibians, indicative of major cross-species transmission events. For example, letoviruses identified from amphibian hosts are highly diverse, with the axolotl letovirus more closely related to viruses in fish and the frog letovirus falling in a more divergent position, forming an outgroup to other viruses in this sub-family (Fig. 3). Indeed, phylogenetic reconciliation analysis found that host-jumping was consistently the most frequent evolutionary event, although there was also clear evidence for host-virus co-divergence that likely reflects deeper branching events in the evolutionary history of these viruses (Fig. 4B).

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Figure 4.

(A) Tanglegram of rooted phylogenetic trees for the Coronaviridae and their hosts. Lines and branches are coloured to represent host class, as in Fig. 2. The ‘untangle’ function was used in TreeMap3.0 to maximise the congruence between the host (left) and virus (right) phylogenies. Supplementary Fig. S2 provides the names of the hosts and viruses. The virus tree is that shown in Fig. 3C. (B) Reconciliation analysis of each virus family using eMPRess (Santichaivekin et al. 2020) illustrates the proportion of specific evolutionary events, with the mean indicated by a red horizontal line. The ‘event costs’ associated with incongruences between trees were conservative towards co-divergence and defined here as follows: 0 for co-divergence, 1 for duplication, 1 for host-jumping and 1 for extinction.

2.4. Amino acid conservation across the Coronaviridae

Compatible with their phylogenetic relationships, the new viruses identified here possessed several motifs that are conserved among the coronaviruses, particularly within the RdRp domain in ORF1b (Fig. 5). These included the amino acid sequences ‘GWDYPKCD’, ‘SGDATTAYANS’, and ‘ILSDDGV’ that are mostly conserved among the Coronaviridae but not the Nidovirales as a whole (Fig. 5). Indeed, these conserved domains are essential for metal ion chelation and binding of the primer 3′-end template complex (Snijder et al. 2003) and have recently been identified as potential ‘barcodes’ for coronavirus identification (Babaian and Edgar 2021).

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Figure 5.

Multiple virus amino acid sequence alignment of the RdRp showing three motifs (A, B and C) conserved throughout all known genera within the Coronaviridae, including the newly discovered viruses within the Letovirinae for which virus transcripts in this region were identified.

3. Discussion

Virological sampling of jawless fish combined with mining publicly available sequence data of bony fish transcriptomes enabled us to identify eight new members of the Nidovirales, six of which fell within the Coronaviridae and a further two fell within the related Tobaniviridae. The six new coronavirus-like transcripts were close genetic relatives of viruses classified within the Letovirinae in the RdRp, including Pacific salmon nidovirus, Microhyla letovirus, and other viruses recently identified in fishes and amphibians, forming a sister clade to all other known coronaviruses.

The discovery of these new viruses not only expands the phylogenetic diversity of the Letovirinae, but it also sheds important new light on the evolution and host range in the Coronaviridae as a whole. In particular, our study revealed that coronavirus hosts include both freshwater and marine fish spanning multiple taxonomic orders. This includes an ancient lineage of jawless fish, herein represented by the pouched lamprey from New Zealand, that represents one of the evolutionary oldest vertebrates still to exist and one that has experienced little morphological change for around 360 million years (Gess, Coates, and Rubidge 2006), although its true age has recently been debated (Miyashita et al. 2021). These jawless and bony fish hosts were also sampled across a range of geographic localities: New Zealand, China, Europe and North America. Although divergent in their sequences, conserved motifs across the entire virus family strongly support the inclusion of these new viruses within the Coronaviridae and more specifically the Letovirinae. With the expansion of virological surveys of wildlife, it is likely that additional viruses within the Coronaviridae will be discovered. Indeed, the long branch lengths separating the divergent families within the Nidovirales are indicative of very limited sampling across this order of RNA viruses.

The genomic structure of both Pacific salmon nidovirus and Ambystoma mexicanum nidovirus includes a short, non-coding region between the ORF1ab and the remaining genes, including the S protein (Edgar et al. 2020; Mordecai et al. 2019). It has been suggested that this homologous gap represents possible genome segmentation in this clade (Edgar et al. 2020). We were, unfortunately, unable to determine the complete genomic structure of the viruses identified here and so cannot directly address this issue. It is noteworthy, however, that the S protein of kanakana letovirus shared more sequence similarity with toroviruses rather than coronaviruses, while trout perch letovirus and Pacific salmon nidovirus consistently fell within the Coronaviridae. This pattern of phylogenetic incongruence between these genomic regions is indicative of past recombination, or reassortment, within the Nidovirales. At present, the occurrence of this process seems to be limited to only some aquatic vertebrate hosts, although given the propensity for coronaviruses to recombine (Denison et al. 2011), it is likely that this will be observed in additional species.

First recorded in 2011, lamprey populations in New Zealand’s Southland region have been affected by LRS, causing reddening along the length of the body with increased mortalities (Brosnahan et al. 2019). While we cannot assign the novel kanakana letovirus discovered here as the causative agent of this syndrome, similar disease symptoms have been observed in salmon infected with Pacific salmon nidovirus (Mordecai et al. 2019). Since lamprey are classed as a nationally vulnerable threatened species (Dunn et al. 2018), it is important that further investigation is undertaken to determine the true prevalence of coronavirus in lamprey populations, whether the virus is associated with LRS and if the virus has made a recent jump from a reservoir host species. The increased deliberate movement of fish around the world for aquaculture, ornamental fish trade, and biofouling on vessels all are pathways for the potential transmission of disease internationally (Cano et al. 2020).

That Letoviruses form a sister clade to all other coronaviruses suggests that the backbone of the Coronaviridae phylogeny overall mirrors that of their vertebrate hosts, with different viral genera associated with different vertebrate classes, and the clear transition from aquatic to land (Zhang et al. 2018). Nevertheless, lamprey represent an ancient lineage of jawless fish that fall sister to all other vertebrates, including jawed fish, amphibians, and reptiles. In this context, it is particularly notable that the novel coronavirus identified here in the pouched lamprey does not fall basal to other fish coronaviruses, in turn revealing that cross-species transmission events have played a major role in the evolutionary history of these viruses and which is also apparent from our co-phylogenetic reconciliation analysis. Hence, as is the case with many families of RNA viruses (Geoghegan, Duchêne, and Holmes 2017), the broad association between different coronaviruses and their vertebrate host classes suggests that the evolutionary history of this group likely dates to at least the origin of the vertebrates some 530 million years ago. However, on top of this overall pattern of long-term virus-host co-divergence, there have also been frequent instances of cross-species virus transmission. As the case of the pouched lamprey demonstrates, such host jumping events may be particularly commonplace in hosts that reside in aquatic environments, perhaps reflecting a combination of evolutionary antiquity (and hence more long-term opportunity for cross-species transmission) and greater physical interactions between multi-species assemblages, although this clearly needs to be investigated in greater detail. Indeed, the parasitic behaviour of lamprey might facilitate viral transmission.

4. Materials and methods

Supplementary Material

Acknowledgements

Contributor Information

Allison K Miller, Department of Anatomy, University of Otago, Dunedin 9016, New Zealand.

Jonathon C O Mifsud, Marie Bashir Institute for Infectious Diseases and Biosecurity, School of Life and Environmental Sciences and School of Medical Sciences, Johns Hopkins Drive, The University of Sydney, Sydney, NSW 2006, Australia.

Vincenzo A Costa, Marie Bashir Institute for Infectious Diseases and Biosecurity, School of Life and Environmental Sciences and School of Medical Sciences, Johns Hopkins Drive, The University of Sydney, Sydney, NSW 2006, Australia.

Rebecca M Grimwood, Department of Microbiology and Immunology, University of Otago, Dunedin 9016, New Zealand.

Jane Kitson, Kitson Consulting Ltd 9 Black Road, Invercargill 9879, Invercargill/Waihopai, New Zealand.

Cindy Baker, National Institute of Water and Atmospheric Research, Hamilton 3216, New Zealand.

Cara L Brosnahan, Animal Health Laboratory and Diagnostic and Surveillance Directorate, Ministry for Primary Industries, Upper Hutt 5018, New Zealand.

Anjali Pande, National Institute of Water and Atmospheric Research, Hamilton 3216, New Zealand. Animal Health Laboratory and Diagnostic and Surveillance Directorate, Ministry for Primary Industries, Upper Hutt 5018, New Zealand.

Edward C Holmes, Marie Bashir Institute for Infectious Diseases and Biosecurity, School of Life and Environmental Sciences and School of Medical Sciences, Johns Hopkins Drive, The University of Sydney, Sydney, NSW 2006, Australia.

Neil J Gemmell, Department of Anatomy, University of Otago, Dunedin 9016, New Zealand.

Jemma L Geoghegan, Department of Microbiology and Immunology, University of Otago, Dunedin 9016, New Zealand. Institute of Environmental Science and Research, 34 Kenepuru Drive, Kenepuru, Porirua 5022 Wellington 5018, New Zealand.

References